MUCILAGE-MODIFIED4 Encodes a Putative Pectin
Biosynthetic Enzyme Developmentally Regulated by
APETALA2, TRANSPARENT TESTA GLABRA1, and
GLABRA2 in the Arabidopsis Seed Coat1
Tamara L. Western2, Diana S. Young, Gillian H. Dean, Wei Ling Tan3, A. Lacey Samuels, and
George W. Haughn*
Botany Department, University of British Columbia, 6270 University Boulevard, Vancouver, British
Columbia, Canada V6T 1Z4
The Arabidopsis seed coat epidermis undergoes a complex process of differentiation that includes the biosynthesis and
secretion of large quantities of pectinaceous mucilage, cytoplasmic rearrangement, and secondary cell wall biosynthesis.
Mutations in MUM4 (MUCILAGE-MODIFIED4) lead to a decrease in seed coat mucilage and incomplete cytoplasmic
rearrangement. We show that MUM4 encodes a putative NDP-l-rhamnose synthase, an enzyme required for the synthesis
of the pectin rhamnogalacturonan I, the major component of Arabidopsis mucilage. This result suggests that the synthesis
of monosaccharide substrates is a limiting factor in the biosynthesis of pectinaceous seed coat mucilage. In addition, the
reduced cytoplasmic rearrangement observed in the absence of a key enzyme in pectin biosynthesis in mum4 mutants
establishes a causal link between mucilage production and cellular morphogenesis. The cellular phenotype seen in mum4
mutants is similar to that of several transcription factors (AP2 [APETALA2], TTG1 [TRANSPARENT TESTA GLABRA1], TTG2
MYB61, and GL2 [GLABRA2]). Expression studies suggest that MUM4 is developmentally regulated in the seed coat by AP2,
TTG1, and GL2, whereas TTG2 and MYB61 appear to be regulating mucilage production through alternate pathway(s). Our
results provide a framework for the regulation of mucilage production and secretory cell differentiation.
The mature seed coat, in addition to forming a
protective layer around the embryo, can play roles in
such processes as seed dispersal and germination.
One adaptation believed to act in these processes is
the production of mucilage in the epidermal cells of
the seed coat. This specialization, known as myxospermy, is found in many families, including the
Brassicaceae, Solanaceae, Linaceae, and Plantaginaceae (Grubert, 1981; Boesewinkel and Bouman,
1995). Seed imbibition in these plants leads to the
formation of a gel capsule around the seed that is
thought to aid hydration and/or dispersal. Mucilage
consists of pectins, a heterogeneous group of acidic
polysaccharides that form a gel-like matrix. In the
primary cell wall, pectins form the matrix in which
cellulose microfibrils and hemicelluloses are embedded. Key pectins include homo-GalUA (HGA) and
1
This work was supported by the Natural Science and Engineering Research Council of Canada (Discovery Grants to G.W.H.
and A.L.S. and a Cell Wall Multi-Network Grant to G.W.H.).
2
Present address: Biology Department, McGill University, 1205
Docteur Penfield Avenue, Montreal, QC, Canada H3A 1B1.
3
Present address: Ottawa Regional Cancer Centre, Centre for
Cancer Therapeutics, Third Floor, 503 Smyth Road, Ottawa, ON,
Canada K1H 1C4.
* Corresponding author; email [email protected]; fax
604 – 822– 6089.
Article, publication date, and citation information can be found
at http://www.plantphysiol.org/cgi/doi/10.1104/pp.103.035519.
296
rhamnogalacturonan I (RGI). HGA consists of an
unbranched chain of ␣-1,4-linked GalUA residues
whose carboxyl groups may be modified through
methyl-esterification. RGI has a backbone of alternating ␣-1,2-linked Rha and ␣-1,4-linked GalUA residues, with sugar side chains attached on varying
numbers of Rha residues. Both HGA and RGI are
believed to be synthesized in the Golgi apparatus
through the activities of glycosyltransferases using
nucleotide sugars imported from the cytoplasm (for
review, see Ridley et al., 2001). Chemical analysis of
Arabidopsis mucilage has shown that it is composed
largely of ␣-1,2 linked Rha and ␣-1,4 linked GalUA,
suggesting that it is comprised primarily of RGI (Penfield et al., 2001). Immunofluorescence studies of mucilage have demonstrated the presence of methylesterified HGA (Willats et al., 2001); however, the
authors did not test for the presence of RGI.
The production of mucilage in the epidermal cells
of the Arabidopsis seed coat is part of a coordinated
developmental process that begins with cell growth,
followed by biosynthesis and polar secretion of large
quantities of pectin, formation of a cytoplasmic column through cytoplasmic constriction and vacuolar
contraction, and, finally, the synthesis of a secondary
cell wall (columella; Beeckman et al., 2000; Western et
al., 2000; Windsor et al., 2000). Seed coat mucilage is
dispensable under laboratory conditions in Arabidopsis and mum (mucilage-modified) mutants affecting
Plant Physiology, January 2004, Vol. 134, pp. 296–306, www.plantphysiol.org © 2004 American Society of Plant Biologists
MUM4 Putative Pectin Enzyme in Seed Coat Differentiation
mucilage production can be identified by a simple
screening method (Western et al., 2001). Mutations in
one of these genes, MUM4, yield a phenotype where
mucilage is not released from hydrated mature seeds.
Several genes encoding putative transcription factors have been implicated in seed coat epidermal
development because mutations in these genes result
in seeds that fail to release mucilage upon hydration.
Mutants in AP2 (APETALA2), in addition to their
defects in floral morphogenesis, lack differentiation
past the growth phase of mucilage secretory cells
(Jofuku et al., 1994; Western et al., 2001). TTG1
(TRANSPARENT TESTA GLABRA1), TTG2, and GL2
(GLABRA2), which were originally identified
through their role in trichome specification, have
defects in both mucilage and columella production in
the seed coat (Koornneef, 1981; Penfield et al., 2001;
Western et al., 2001; Johnson et al., 2002). It has been
shown at the genetic and molecular levels in
trichomes and root hairs that TTG1 interacts with a
basic helix-loop-helix (bHLH) protein and a tissuespecific MYB protein to activate both GL2 and TTG2
(Payne et al., 2000; Johnson et al., 2002; Schiefelbein,
2003). Recently, mutations in MYB61 have been
found to specifically affect both mucilage and columella production in the Arabidopsis seed coat (Penfield et al., 2001).
The objective of this study was to characterize the
role of MUM4 in the development of the Arabidopsis
seed coat. Using positional cloning of MUM4, we
demonstrate that MUM4 encodes a putative NDP-lRha synthase. Mutations in this gene lead to reduced
mucilage in the seed coat and an altered columella.
Expression studies show that MUM4 is developmentally regulated during seed coat differentiation such
that its transcript levels are increased at the time of
mucilage production. Furthermore, MUM4 appears
to be a downstream target of a cascade of transcription factors that includes AP2, TTG1, and GL2. These
results demonstrate the importance of mucilage production for the morphology of seed coat epidermal
cells and suggest a regulatory framework for the
control of seed coat epidermal differentiation.
RESULTS
mum4 Mutant Phenotype
Two alleles of mum4 (mum4-1 and mum4-2) were
identified in a screen for mutants that lacked extruded mucilage as visualized by staining with Ruthenium red (compare Fig. 1, A with D; Western et
al., 2001). A T-DNA insertion allele, mum4-3, was also
identified. Because all alleles of this mutant had similar phenotypes, only that of mum4-1 is described.
Viewed with scanning electron microscopy, mature
mum4 seed coats are marked not only by the absence
of mucilage extrusion upon hydration but also by the
absence of volcano-shaped columellae (compare Fig.
1, B with E). Light microscopy of sections of mature
Plant Physiol. Vol. 134, 2004
seeds revealed that both columellae and mucilage are
present in mum4 but reduced in comparison with
wild type (compare Fig. 1, C with F). No other phenotypes were observed.
To more fully characterize the mum4 seed coat
epidermal cell defects, their development was studied with light microscopy (Fig. 1, G–L). Under our
growth conditions, seed maturation to dry seed takes
approximately 18 d and has been staged by DPA. In
terms of embryo growth, 4 DPA is early to mid
globular stage, 7 DPA is mid-torpedo stage, whereas
10 DPA is late upturned-U stage (this study; Western
et al., 2000). The early stages of mum4 epidermal cell
differentiation are similar to that of wild type: The
cells grow, and mucilage starts to accumulate, accompanied by the initiation of intracellular cytoplasmic rearrangement to form a column in the center of
the cell (compare wild type, Fig. 1, G and H, with
mum4, Fig. 1, J and K). Differences, however, were
obvious at 10 DPA. Although wild-type seed coat
cells have dark-pink staining mucilage in the extracellular space and a thin secondary cell wall surrounding a central column of cytoplasm (Fig. 1I), the
extracellular space containing mucilage in mum4 seed
coat epidermal cells is smaller with lighter staining
mucilage and the secondary cell wall has formed
around a partially formed cytoplasmic column atop a
large vacuole (Fig. 1L). Thus, the seed coat appears to
have less mucilage, and the flattened columella appears to be the result of reduced cytoplasmic and
vacuolar constriction.
The possibility of a change in mucilage quantity or
composition in mum4 versus wild-type seed coats
was addressed by quantifying monosaccharides using gas chromatography. Because mum4 seed coats
release mucilage in the presence of ammonium oxalate, mucilage could be extracted from intact mum4
seeds for direct comparison with wild-type mucilage.
Trimethylsilane (TMS) derivatives were used because this method allows detection of GalUA and
neutral sugars simultaneously. The results showed
that, for equal seed masses, less sugar was extracted
from mum4 mucilage, suggesting that less mucilage is
synthesized compared with wild-type seeds (Fig. 2).
In addition, the decrease in monosaccharides in
mum4 mucilage was limited primarily to two sugars,
GalUA and a single neutral sugar (identified as either
Rha or Fuc; Fig. 2; H0 ␮1 ⫽ ␮2, GalUA, T ⫽ 7.897, P ⬍
0.01; Rha, T ⫽ 6.960, P ⫽ 0.01). Because the identity of
the neutral sugar could not be completely resolved
using TMS derivatives, alditol acetate derivatization,
which gives a single derivative for each monosaccharide, was used to confirm that it is Rha (Fuc found
only at trace levels in wild-type mucilage; data not
shown). These results are consistent with the suggestion that Arabidopsis mucilage is comprised primarily of relatively unbranched RGI (Penfield et al.,
2001).
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Figure 1. Structure and development of wild-type (Columbia-2 [Col-2]) and mum4-1 seed coats. A, Whole-mount wild-type
seed stained with Ruthenium red. Note red staining capsule of mucilage surrounding seeds. B, Scanning electron micrograph
of wild-type seed coat. Note thickened radial cell walls (arrow) and central columella (arrowhead) in each cell. C, Cross
section of mature wild-type seed coat epidermis. The cells have burst to release mucilage, leaving cell wall fragments
attached to tip of columellae (arrow). D, Whole-mount mum4-1 seeds stained with Ruthenium red. No mucilage is visible
around seeds. E, Scanning electron micrograph of mum4-1 seed coat. Columellae are not visible. F, Cross section of mature
mum4-1 epidermal cells. Flattened columellae (arrow) are apparent and intact primary cell walls (arrowhead) surround faint
pink staining mucilage. G to I, Cross sections of developing wild-type epidermal cells. G, 4 DPA. H, 7 DPA, note
accumulation of pink-staining mucilage in cells. I, 10 DPA, the center of each cell contains cytoplasm that is surrounded
by a developing secondary cell wall staining blue (arrow), whereas both are surrounded by intensely stained mucilage (dark
pink). J to L, Developing mum4-1 epidermal cells. J, 4 DPA. K, 7 DPA, pink staining mucilage is only apparent in top portion
of cells. L, 10 DPA, the cytoplasm fills most of the cell in a dome shape, with a thin, blue secondary cell wall (arrow) forming
around it. Pink staining mucilage is found only in the upper corners of the cells. C and F to L, Toluidine blue-stained sections
of tissue fixed under aqueous conditions. Scale bars: A and D ⫽ 100 ␮m, B and E ⫽ 20 ␮m, and C and F to L ⫽ 10 ␮m.
Arabidopsis Mucilage Is Largely Composed of RGI
Figure 2. GalUA and Rha levels of wild-type (Col-2) and mum4-1
mucilage. Comparison of GalUA and Rha levels detected in mucilage isolated from intact mum4-1 seeds (white bars) versus wild-type
mucilage (black bars, Col-2) using TMS derivatization and gas chromatography. The bars represent micrograms of sugar per milligram of
seed as determined by comparison with an internal standard. Analyses were done in triplicate. Error bars ⫽ SD.
298
To further investigate the effect of mum4 mutations
on mucilage production, immunofluorescence with
antipectin antibodies was used. Whole-mount immunofluorescence performed on wild-type Arabidopsis
seeds using the polyclonal antipectin antibody
␣-poly-GalUA (PGA)/RGI gave significant labeling
of the mucilage without pretreatment (dry seeds
added directly to fixative; Fig. 3A). This polyclonal
antibody, although raised to RGI, has some crossreactivity to HGA (Lynch and Staehelin, 1992). However, JIM5 and JIM7 monoclonal antibodies specific
for HGA with moderate or higher states of methylation, respectively, failed to cross-react with the mucilage under the same conditions (Fig. 3C; data not
shown). Slight (JIM5; data not shown) or moderate
(JIM7; Fig. 3D) amount of labeling of extruded mucilage could be observed only after a pretreatment of
Plant Physiol. Vol. 134, 2004
MUM4 Putative Pectin Enzyme in Seed Coat Differentiation
tioned, and then labeled by indirect immunofluorescence. Labeling was seen on thick sections of developing wild-type seed coats stained with ␣-PGA/RGI
(9 DPA; Fig. 3E), with fluorescence in the epidermal
cells on either side of a central, unstained space.
Control experiments including omitting the primary
antibody or substituting nonimmune, normal rabbit
serum for the primary antibody, abolished the fluorescent signal (Fig. 3F). The similarity in staining
pattern with ␣-PGA/RGI in sections compared with
the pink staining substance in toluidine blue-stained
sections (e.g. Fig. 1I) confirms the identity of the pink
staining substance as pectinaceous mucilage. When
thick sections of mum4 seeds were treated with
␣-PGA/RGI, only a small amount of labeling was
observed on the outer edges of the epidermal cells
(Fig. 3H) compared with wild-type seeds (Fig. 3G),
once again suggesting a reduced amount of mucilage
in mum4 seeds.
Molecular Cloning of MUM4
Figure 3. Immunofluorescence of wild-type (Col-2) and mum4-1
mucilage with antipectin antibodies. A, Immersion immunofluorescence of whole wild-type seeds, labeled with ␣-PGA/RGI primary
and anti-rabbit Alexa 594 secondary antibody. Note autofluorescence of seed itself and stain of mucilage capsule surrounding the
seed. B, Immersion immunofluorescence of whole mum4-1 seed,
labeled with ␣-PGA/RGI after shaking in ammonium oxalate. The
stained mucilage capsule is thin compared with wild type. C and D,
Immersion immunofluorescence of whole wild-type seeds, labeled
with JIM7 as primary antibody, followed by anti-rat fluorescein isothiocyanate secondary antibody. C, Seed treated as in A with no
shaking pretreatment. No stain of mucilage capsule. D, Seed pretreated by imbibition of extensive shaking in water. Stain of mucilage
capsule is apparent. E to H, Immunofluorescence of sections of
9-DPA seeds incubated with ␣-PGA/RGI or serum control. E, Wildtype seed incubated with ␣-PGA/RGI antibody, epidermal cells are
labeled on seed surface. F, Wild-type seed incubated with nonimmune normal rabbit serum control, no staining of seed epidermis,
same photographic conditions as E. G, Direct comparison of wildtype seed and H mum4-1 seed show reduced ␣-PGA/RGI label in
mum4 mutant. Scale bars: A to F ⫽ 100 ␮m and G and H ⫽ 50 ␮m.
imbibition with extensive shaking before fixation.
These results suggest that most of the binding found
against wild-type mucilage can be attributed to RGI,
rather than HGA. When mum4 seeds were pretreated
by shaking in ammonium oxalate, ␣-PGA/RGI labeling of a thin ring around the seed was observed (Fig.
3B), suggesting less mucilage is produced in mum4
seeds compared with wild type.
To examine the mucilage in situ, samples were
cryo-fixed/freeze substituted, resin embedded, secPlant Physiol. Vol. 134, 2004
To identify why mum4 seeds had reduced mucilage, we cloned MUM4 by map-based cloning.
MUM4 was mapped to a 144-kb region spanning the
bacterial artificial chromosomes F12M16 and T3F20.
To identify the exact locus, the mum4-3 T-DNA insertion allele was probed with sequences derived
from these bacterial artificial chromosomes, and an
insertion was identified in a region of T3F20 containing four predicted genes. PCR performed using genespecific primers in combination with a T-DNA right
border primer led to the identification of a T-DNA
insertion in the predicted gene At1g53500.
Next, we isolated a putative full-length cDNA by
reverse transcriptase (RT)-PCR using a series of
primers upstream from the predicted translation
start site. Sequencing of a putative full-length
At1g53500 transcript revealed that the gene consists
of three exons and two introns (including one in the
5⬘-untranslated region [UTR]) and encodes a transcript of 2,477 bp (Fig. 4A).
To confirm that At1g53500 is MUM4, we sequenced
this locus in mum4-1, mum4-2, and mum4-3. All three
alleles contain mutations in the second exon. mum4-1
and mum4-2 contain point mutations leading to G to
A transitions at nucleotides 595 and 886, respectively
(Fig. 4A). These missense mutations should result in
a conserved Asp residue being replaced with an Asn
in mum4-1 and a Gly being replaced with an Arg in
mum4-2 (Fig. 5). mum4-3 contains a T-DNA insertion
at nucleotide 1,637 (Fig. 4A). The similar phenotype
between the missense alleles and that of the mum4-3
insertional allele that lacks detectable transcript (data
not shown) suggests that these changes have a significant effect on protein activity. A database search
of the Salk Institute sequence-indexed Arabidopsis
T-DNA insertion lines identified two new alleles
of mum4, mum4-4, and mum4-5 (Salk_085051 and
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Western et al.
served amino acids (Fig. 5) in the N-terminal, putative NDP-d-Glc 4,6-dehydratase domain.
A comparison with other Arabidopsis genes predicted from genome annotation revealed that MUM4
is a member of a small gene family of putative nucleotide sugar interconversion factors that was initially described in an in silico analysis by Reiter and
Vanzin (2001). The gene family consists of three
genes, RHM1 (At1g78570), RHM2 (MUM4) and
RHM3 (At3g14790), which are 83% to 89% identical at
the amino acid level (Fig. 5). This suggests that there
are at least three genes with a similar function.
Expression Pattern of MUM4
The expression pattern for MUM4 mRNA in different tissues was determined qualitatively using RTFigure 4. Structure of the MUM4, RHM1, and RHM3 genes and
complementation of mum4-1 with MUM4 coding region. A, Exonintron structures are based on comparison of the genomic and cDNA
sequences. The coding region is shown as black boxes, whereas the
gray boxes represent 5⬘- and 3⬘-UTRs. The location of the two
domains with similarity to bacterial nucleotide sugar interconversion
enzymes are represented as thin bars underneath the MUM4 coding
region. ⱍ, Location of the two EMS alleles (mum4-1 and mum4-2); ∨,
Location of the three T-DNA insertion alleles (mum4-3, mum4-4, and
mum4-5). B, Complementation of mum4-1 with pMUM4g. Mucilage
capsule is observed around seed stained with Ruthenium red. C,
mum4-1 seed transformed with empty vector pGREEN0229. No
mucilage release is observed. Scale bars ⫽ 100 ␮m in B and C.
038898, respectively), both of which also have insertions in the second exon (Fig. 4A). Based on the
Ruthenium red assay, both of these mutants have a
phenotype (data not shown) similar to mum4-1 (Fig.
1D). The wild-type At1g53500 sequence plus 2.1 kb
upstream and 0.4 kb downstream sequence was
cloned to give the plasmid pMUM4g. Transformation
of mum4-1 plants with pMUM4g versus an empty
vector control showed that the wild-type At1g53500
genomic clone could rescue the mucilage phenotype
(Fig. 4, B and C). Together, these results demonstrate
that At1g53500 is MUM4.
MUM4 encodes a protein of 667 amino acids (Fig.
5). Comparison with GenBank sequences suggested
that MUM4 contains two domains: an N-terminal
domain with similarity to bacterial dTDP-d-Glc-4,6dehydratases (44% identical, 61% similar to RfbB
[COG1088] consensus) and a C-terminal domain with
some similarity to bacterial 4-reductases (20% identical, 34% similar to RfbD [COG1091] consensus) (Tatusov et al., 2000; Figs. 4A and 5). Both domains
contain the conserved N-terminal NAD⫹ binding
(GxxGxxG) and active site catalytic couple (YxxxK)
motifs found in members of the reductase/epimerase/dehydrogenase protein superfamily (Graninger
et al., 1999; Allard et al., 2001). Both of the missense
alleles, mum4-1 and mum4-2, change codons of con300
Figure 5. Alignment of the amino acid sequence of the MUM4
protein with those of RHM1 and RHM3. The putative 4,6dehydratase and 4-reductase domains within MUM4 are marked
with single and double underlines, respectively, whereas the highly
conserved NAD(P)⫹ cofactor-binding (GxxGxxG) and active site catalytic couple (YxxxK) motifs are indicated above the sequence alignment. The amino acid substitutions (mum4-1 and mum4-2) and sites
of T-DNA insertion (mum4-3, mum4-4, and mum4-5) are also
marked.
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MUM4 Putative Pectin Enzyme in Seed Coat Differentiation
PCR at a saturating cycle number. As suggested by
expressed sequence tag data, MUM4 transcripts are
apparent in all tissues tested: stems, roots, leaves,
seedlings, inflorescence tips, and siliques (Fig. 6A). A
similar analysis was done for RHM1 and RHM3. As
with MUM4, both RHM1 and RHM3 transcripts were
detected in all tissues (Fig. 6A). RHM3, however,
unlike MUM4 and RHM1, appeared to have only low
expression in some tissues (leaves and inflorescences) because bands for these tissues were not saturated in several trials, even at the high cycle number
used for qualitative RT-PCR.
Because the only obvious phenotype in mum4
plants is the defect in the seed coat epidermis, we
looked at the expression of MUM4 during seed coat
differentiation (Fig. 6B). RNA was extracted from
siliques at three stages of development: (a) before
mucilage synthesis (4 DPA; see Fig. 1G), (b) midmucilage synthesis (7 DPA; Fig. 1H), and (c) after the
completion of mucilage synthesis (10 DPA; Fig. 1I),
and subjected to northern analysis. This experiment
showed that although MUM4 is expressed through-
Figure 6. MUM4, RHM1, and RHM3 expression in Arabidopsis
plants. A, Saturating, qualitative RT-PCR of RNA isolated from wildtype (Col-2) stem, root, leaf, seedling, inflorescence, and siliques
using gene-specific primers for MUM4, RHM1, and RHM3. The
loading control is GAPC, which encodes cytosolic glyceraldehyde3-phosphate dehydrogenase. B, Changes in MUM4 expression during silique development. RNA gel-blot analysis using MUM4 probe
on total RNA from wild-type (Col-2) inflorescence, 4, 7, and 10 DPA
wild-type siliques, and 7 DPA ap2-7 siliques. Ethidium bromidestained rRNA is shown as loading control.
Plant Physiol. Vol. 134, 2004
out seed coat differentiation, transcript levels are
highest at the time of mucilage synthesis (7 DPA; Fig.
6B). To test if the higher levels of MUM4 detected at
7 DPA may be largely restricted to the seed coat,
ap2-7 mutants were used as a control in gel blots (Fig.
6B). ap2 was chosen because the outer two layers of
the seed coat of ap2 mutant seeds fail to differentiate,
whereas all other cell types of the seed (embryo,
endosperm, and seed coat endothelium) appear normal (Western et al., 2001). The low level of MUM4
transcript seen in 7-DPA ap2-7 siliques suggests that
a large proportion of the MUM4 transcript at 7 DPA
in wild-type siliques may be specific to the outer
layers of the seed coat.
Maximal Accumulation of MUM4 Transcript Requires
Upstream Transcription Factors
Mutations in AP2, TTG1, TTG2, GL2, and MYB61,
which encode putative transcription factors, lead to
seeds with reduced mucilage in the seed coat. Furthermore, developmental analyses have shown that
the cellular phenotypes for ttg1, gl2, and myb61 mucilage secretory cells are almost identical to those of
mum4 mutants, whereas the ap2 mutant cellular phenotype is more severe (Penfield et al., 2001; Western
et al., 2001). Therefore, we tested whether the accumulation of MUM4 transcript was altered in mutants
for each of these genes. RNA was extracted from 7
DPA siliques of wild type and mutants and subjected
to northern hybridization (Fig. 7A). To control for
ecotype differences between mutants isolated in
Col-2 (ap2-7 and myb61-1) versus Ler (ap2-1, gl2-1,
ttg1-1, and ttg2-1) backgrounds, both wild types were
examined. Interestingly, MUM4 appeared to be more
highly expressed in the Ler background, possibly
because of background mutations affecting morphological differences in the seeds and/or siliques between the two ecotypes. As mentioned above, the
level of MUM4 transcript was reduced in ap2-7 mutant siliques compared with wild-type Col-2 siliques,
a result that was confirmed with ap2-1 in the Ler
background. Our analysis also indicated that the levels of MUM4 RNA at 7 DPA are lower for ttg1-1 and
gl2-1 siliques (Fig. 7A), suggesting that AP2, TTG1,
and GL2 each are required for maximum levels of
MUM4 expression at the time of mucilage production. Conversely, there was a slight increase in
MUM4 transcript levels in myb61-1 and ttg2-1 siliques compared with wild type (Fig. 7A). Thus, the
requirement of MYB61 and TTG2 for mucilage biosynthesis is for some aspect other than the regulation
of MUM4 transcription.
The Expression of Putative Regulatory Transcription
Factors during Mucilage Secretory Cell Differentiation
The ap2 mutant phenotype differs from that of ttg1,
ttg2, gl2, and myb61 in that the differentiation of the
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Western et al.
TTG1 has been shown to be required for maximal
expression of both TTG2 and GL2 in leaves (Johnson
et al., 2002; Schiefelbein, 2003). To determine if the
same is true in seed coats, the expression of both GL2
and TTG2 were tested in ttg1-1 compared with wildtype Ler 7 DPA siliques using semiquantitative RTPCR. The transcript levels for both GL2 and TTG2
were reduced in ttg1-1 mutants (Fig. 7C), suggesting
that TTG1 also acts upstream of GL2 and TTG2 in the
seed coat.
DISCUSSION
Figure 7. Expression of MUM4 and genes encoding putative regulatory transcription factors during silique development. A, RNA gelblot analysis using MUM4 probe on total RNA from 7-DPA wildtype, ap2-7, myb61-1 (Col-2), wild-type, ap2-1, gl2-1, ttg1-1, and
ttg2-1 (Landsberg erecta [Ler]) siliques. Ethidium bromide stained
rRNA is shown as loading control. B, Semiquantitative RT-PCR amplification of RNA isolated from wild-type and ap2-7 (Col-2) siliques
at 4 and 7 DPA using gene-specific primers for AP2, TTG1, TTG2,
MYB61, and GL2. The loading control is GAPC, which encodes
cytosolic glyceraldehyde-3-phosphate dehydrogenase. C, Semiquantitative RT-PCR amplification of RNA isolated from wild-type and
ttg1-1 (Ler) 7-DPA siliques using gene-specific primers for TTG2 and
GL2. The loading control is GAPC, which encodes cytosolic
glyceraldehyde-3-phosphate dehydrogenase.
mucilage secretory cells is halted before mucilage
and columella production in ap2 whereas the others
resemble mum4, having low levels of mucilage and
flattened columellae. Thus, phenotypically, AP2 appears to be working upstream of the other genes. To
determine whether AP2 acts by controlling the transcription of the other genes, semiquantitative RTPCR was used to determine the levels of TTG1, TTG2,
GL2, and MYB61 in ap2-7 mutants versus wild type at
4 and 7 DPA. The results showed a significant decrease in transcript levels for GL2 at both 4 and 7
DPA (Fig. 7B) and TTG2 at 4 DPA (TTG2 levels at 7
DPA too low to differentiate between wild type and
ap2; Fig. 7B). This result suggests that AP2 is required
for maximal levels of both GL2 and TTG2 expression
during seed coat differentiation.
302
The development of the mucilage secretory cells of
the Arabidopsis seed coat is a complex process involving the biosynthesis and secretion of a large
quantity of pectinaceous mucilage, intracellular cytoplasmic rearrangement, and secondary cell wall production. We have shown that the putative NDP-lRha synthase encoded by MUM4 is required for
wild-type levels of mucilage biosynthesis and columella formation. Expression of MUM4 is upregulated during seed coat differentiation and is regulated by several transcription factors required for
seed coat epidermal cell differentiation. These data
validate the utility of the mucilage secretory cells as
a model system for polysaccharide biosynthesis, establish a regulatory framework for seed coat epidermal differentiation, and provide a causal link between pectin biosynthesis and cell morphogenesis.
MUM4 Encodes a Putative NDP-L-Rha Synthase
Our data support the hypothesis that MUM4 encodes an enzyme involved in RGI biosynthesis. First,
analysis of mum4 mucilage using antibodies and gas
chromatography have demonstrated a significant reduction in RGI and its composite backbone monosaccharides Rha and GalUA in comparison with wildtype seeds. Second, cloning of MUM4 revealed a
putative protein containing similarity to bacterial nucleotide sugar interconversion enzymes, suggesting
that it, too, is required for the production of activated
sugars. The N-terminal portion of MUM4 is most
similar to bacterial dTDP-d-Glc 4,6-dehydratases, the
first of three enzymes required for the conversion of
dTDP-d-Glc to dTDP-l-Rha (Tonetti et al., 1998).
These data favor a role for MUM4 in the synthesis of
NDP-l-Rha, a key step in the production of RGI. A
reverse genetics approach taken by another group has
come to a similar conclusion regarding the putative
enzymatic function of MUM4 (also designated RHM2
[RHAMNOSE BIOSYNTHESIS 2]; B. Usadel et al.,
2004). However, another role in RGI biosynthesis cannot be ruled out without evidence of enzymatic activity in the conversion of NDP-d-Glc to NDP-l-Rha.
Gram-negative bacteria such as Escherichia coli encode three separate enzymes (4,6-dehydratase, 3,5epimerase, and 4-reductase) to convert dTDP-d-Glc
Plant Physiol. Vol. 134, 2004
MUM4 Putative Pectin Enzyme in Seed Coat Differentiation
to dTDP-l-Rha (Tonetti et al., 1998). The putative
MUM4 protein not only contains an N-terminal domain with similarity to the bacterial dTDP-d-Glc 4,6dehydratases but also a C-terminal domain with
some similarity to 4-reductases (this study; Reiter
and Vanzin, 2001). There is a precedent in Arabidopsis for a bifunctional 3,5 epimerase, 4-reductase in the
synthesis of GDP-l-Fuc from GDP-d-Man (GER1,
GER2) (Bonin and Reiter, 2000). Therefore, based on
sequence analysis, it is attractive to postulate a single,
multifunctional protein acting in the conversion of
NDP-d-Glc to NDP-l-Rha in Arabidopsis (Reiter and
Vanzin, 2001).
MUM4 is a member of a small gene family consisting of three genes (RHM1, MUM4/RHM2, and RHM3;
Reiter and Vanzin, 2001) that exhibit high identity at
both nucleotide and amino acid levels. The ubiquitous expression of RHM1 and RHM3 can be postulated to provide a mechanism for the formation of the
normal primary cell wall in mum4 mutants and the
residual mucilage present in mum4 seed coats. Redundancy in genes coding for putative NDP-l-Rha
synthases is unsurprising because of the importance
of the Rha-containing pectins RGI and RGII in primary cell walls. In fact, the presence of multiple
genes coding enzymes involved in nucleotide sugar
interconversions is common in plants (Reiter and
Vanzin, 2001). The roles of MUM4 and its paralogs
may not be completely redundant, however, because
the three genes do not have identical patterns/levels
of expression throughout all plant tissues (Fig. 6A;
Arabidopsis expressed sequence tag database at The
Institute for Genomic Research [http://www.tigr.
org]; T.L. Western and G.W. Haughn, unpublished
data).
pathways controlling mucilage biosynthesis in the
seed coat. In one of these pathways, it appears that
TTG1 and GL2 control the transcription of MUM4
(Fig. 8). In other epidermal systems, such as
trichomes and root hairs, TTG1 has been shown to
interact with a tissue-specific MYB protein through
the bHLH protein GLABRA3 (Payne et al., 2000;
Schiefelbein, 2003) to activate GL2. Therefore, we
favor a model in which a seed coat-specific complex
of TTG1-bHLH-MYB causes activation of GL2 and
the subsequent up-regulation of MUM4 during mucilage production (Fig. 8). Recent results suggest that
EGL3 (ENHANCER OF GLABRA3) and/or TT8
(TRANSPARENT TESTA8) are the bHLH proteins
acting with TTG1 in the seed coat (Zhang et al., 2003).
A possible MYB component of this trimeric complex,
MYB23, is expressed in developing seeds and groups
to the same subfamily of MYBs as the bHLH-TTG1
interacting proteins GLABROUS1 and WEREWOLF
(Kirik et al., 2001).
Correct regulation of MUM4 transcription at the
time of mucilage production does not require TTG2
or MYB61 (Fig. 7A). However, TTG1 is required to
activate TTG2 in the seed coat (Fig. 7C) and in other
tissues (Johnson et al., 2002). This result places TTG2
downstream from TTG1 in a second pathway that
controls mucilage biosynthesis in the developing
seed. This alternate pathway, along with another that
involves MYB61, may regulate the expression of
other proteins involved in RGI synthesis including an
NDP-d-GlcUA 4-epimerase (Feingold, 1982; Reiter
and Vanzin, 2001), a nucleotide sugar transporter or
an RGI-backbone glycosyltransferase. Alternatively,
MYB61 and TTG2 may be involved in the transport of
newly synthesized RGI to the plasma membrane.
Feedback inhibition of mucilage production in the
Developmental Regulation of MUM4 during Seed Coat
Secretory Cell Differentiation by AP2, TTG1, and GL2
MUM4 transcript increases in differentiating siliques at the time of mucilage production. Two lines
of evidence suggest that this up-regulation occurs in
the seed coat epidermis to support mucilage biosynthesis. First, the only obvious phenotypic defect in
mum4 plants occurs in the seed coat epidermis. Second, MUM4 expression is severely attenuated in siliques of ap2 mutants that fail to differentiate the
outer two layers of the seed coat. Such a specific
up-regulation of a putative NDP-l-Rha synthase may
be required to provide extra Rha for the production
of the large quantity of RGI required for mucilage
synthesis. If so, the amount of this enzyme must be
the limiting factor in Rha biosynthesis and the
amount of Rha a limiting factor in RGI biosynthesis.
Our data indicate that MUM4 transcription is decreased in ttg1 and gl2 mutants but is essentially wild
type in ttg2 and myb61 mutants (Fig. 7A). This result
suggests a regulatory framework for mucilage secretory cell differentiation in which there are at least two
Plant Physiol. Vol. 134, 2004
Figure 8. Proposed genetic pathway for the regulation of mucilage
production during seed coat secretory cell differentiation. AP2 and a
TTG1 complex with a bHLH protein (candidates include EGL3
and/or TT8) and a tissue-specific MYB transcription factor activate
GL2 and TTG2. GL2 acts upstream of MUM4. In contrast, both TTG2
and MYB61 appear to affect aspects of mucilage production independent from MUM4. For details, see text.
303
Western et al.
absence of secretion has been demonstrated in the
root caps of the maize (Zea mays) mutant Ageotropic
(Millar and Moore, 1990).
AP2 encodes a putative transcription factor that is
required for the differentiation of the outer two layers of the seed coat (Western et al., 2001). Not surprisingly, ap2 seeds lack mucilage and fail to activate
MUM4 transcription beyond its baseline level of expression (Fig. 7A). Although AP2 is required for maximum GL2 and TTG2 transcript levels (Fig. 7B), transcription of TTG1 and MYB61 was independent of
AP2 activity (Fig. 7B). Our data are consistent with
the hypothesis that AP2 functions in parallel with
TTG1 to activate GL2 and TTG2 in the seed coat
epidermis (Fig. 8).
Columella Production during Seed Coat Secretory
Cell Differentiation
The mum4 phenotype is characterized by both a
reduction in the amount of mucilage produced and
by a flattened columella in the seed coat epidermis.
The flattened appearance of the columella in mum4
seeds appears to be the result of incomplete cytoplasmic rearrangement and vacuole constriction, leaving
a dome of cytoplasm over which the secondary cell
wall is laid. Although the defect in mucilage production is readily explained by the putative function of
MUM4 as an NDP-l-Rha synthase, the connection
with the columella shape change is less obvious. At
least two hypotheses can explain the dependence of
cell morphogenesis on mucilage synthesis. The formation of the apoplastic space and cytoplasmic column may be driven by the pressure of accumulating
mucilage in the extracytoplasmic space and/or the
formation of an osmotic gradient between the cell
and the hydrophilic pectin may lead to the loss of
turgor and size reduction of the vacuole. Such passive processes may contribute to cell morphogenesis
but seem too simple to account for the apparent
complexity of the shape of the cytoplasmic column.
An alternate but not mutually exclusive explanation
is that the synthesis of mucilage may represent or
lead to the production of an oligosaccharide signal
that stimulates the cell to undergo morphogenesis
(Dumville and Fry, 2000; Ridley et al., 2001). Determination of the exact processes involved would be
facilitated by the isolation of mutants affected specifically in the shaping of the columella.
MATERIALS AND METHODS
Plant Material and Growth Conditions
Lines of Arabidopsis used were mum4-1, mum4-2 (Col-2 ecotype; Western
et al., 2001), mum4-3 (see below), mum4-4, mum4-5 (Salk_085051 and 038898,
respectively, obtained from the Arabidopsis Biological Resource Center
[ABRC], Ohio State University, Columbus), ap2-1 (Ler ecotype; ABRC), ap2-7
(Col-2; Kunst et al., 1989), gl2-1, ttg1-1, ttg2-1 (Ler; ABRC), and myb61-1
(Col-0; gift from Michael Bevan, John Innes Centre, Norwich, UK). Growth
conditions were as described by Western et al. (2001).
304
mum4-3 was isolated by screening T-DNA mutagenized lines (Feldmann
and Marks, 1987; ABRC) by staining with a 0.01% (w/v) aqueous solution of
Ruthenium red.
Staging of Flower Age
Flowers were staged as by Western et al. (2001).
Immunofluorescence
Developing seeds for bright-field microscopy were prepared as described
by Western et al. (2000).
For immunofluorescence, developing seeds were placed in copper hats in
a 0.2 m Suc solution and high-pressure frozen using a Balzers HPM 101
(Balzers Instruments, Balzers, Liechtenstein). The samples were freeze substituted over 5 d in 0.25% (v/v) glutaraldehyde/8% (v/v) dimethoxypropane in acetone using a dry ice/acetone bath at ⫺80°C, then gradually
warmed to ⫺20°C, 4°C, and then 20°C (3 h at each temperature) before
embedding in LR White resin. Sectioning and microscopy were performed
as for bright field.
Immersion immunofluorescence was performed according to the procedure of Willats et al. (2001), except dry wild-type seeds were placed directly
into the fixative. Alternatively, the seeds were imbibed for 24 h in buffer
with gentle shaking before fixation, all subsequent steps also being performed with shaking. Antipectin monoclonal antibodies JIM5, JIM7, and
polyclonal ␣-PGA/RGI at 1:5 and 1:10 (v/v) dilutions were used as primary
antibodies. Pre-immune serum controls were rat (JIM5 and JIM7) and rabbit
(␣-PGA/RGI). Secondary antibodies were 1:100 (v/v) dilutions in 5% (w/v)
nonfat dry milk (NFDM) of goat anti-rat IgG conjugated to fluorescein
isothiocyanate (JIM5 and JIM7) and anti-rabbit IgG (␣-PGA/RGI). Sections
were mounted in either anti-fade agent (JIM5 and JIM7; Slowfade Antifade,
Molecular Probes, Eugene, OR) or 1:2 (v/v) glycerol:water (␣-PGA/RGI)
before observation.
For immunofluorescence, 0.5-␮m sections were incubated in 50 mm
EGTA for 1 h, blocked with 5% (w/v) NFDM for 20 min, then incubated for
1 h with primary antibodies at 1:5 and 1:10 (v/v) dilutions in 5% (w/v)
NFDM, and for 1 h in secondary antibodies diluted at 1:100 (v/v). Rinses
were performed before and after each incubation with 1⫻ Tris-buffered
saline/0.1% (v/v) Tween 20. Primary antibodies, secondary antibodies,
pre-immune controls, and mounting were done as described for immersion
immunofluorescence.
Scanning Electron Microscopy
Dry-mounted seeds were prepared and observed as by Western et al.
(2001).
Gas Chromatography
Mucilage was extracted from samples of approximately 5 mg of intact
seeds by shaking in 0.2% (w/v) ammonium oxalate for 2 h at 30°C. No
significant difference in mass was observed between Col-2 and mum4-1 seed
(four lots of 100 counted seed weighed in triplicate; Col-2 ⫽ 2.0 ⫾ 0.3 mg,
mum4-1 ⫽ 2.2 ⫾ 0.1 mg sd). Ten microliters of internal standard (5 mg mL⫺1
myo-inositol) was added before precipitation with 5 volumes of absolute
ethanol and drying under nitrogen gas. Derivatization, gas chromatography, and identification of trimethylsilyl ethers were performed as described
previously (Western et al., 2000).
For alditol acetates, samples were hydrolyzed in trifluoroacetic acid for
2 h at 100°C, dried under nitrogen gas, then reduced for 90 min at 40°C in
245 ␮L of 2.6 m ammonia and 700 ␮L of 2% (w/v) sodium borohydride in
dimethylsulfoxide. After addition of 175 ␮L of acetic acid, the samples were
acetylated for 10 min at room temperature with 175 ␮L of
1-methylimidazole and 2.8 mL of acetic anhydride. Water (5.6 mL) was
added, and the alditol acetates were extracted with 1.05 mL of dichloromethane (DCM). The recovered phase was dried and resuspended in 200
␮L of DCM, then re-extracted with 1 mL of water. This recovered DCM
phase was used for analysis. Samples were run on a gas chromatograph
(model 5890A, Hewlett-Packard, Mississauga, ON, Canada) on a HP-23
glass capillary column (30-m ⫻ 0.25-mm i.d.) with helium as the carrier gas.
The temperature program was 2 min at 180°C, increase 10°C min⫺1 up to
Plant Physiol. Vol. 134, 2004
MUM4 Putative Pectin Enzyme in Seed Coat Differentiation
200°C, 5 min at 200°C, increase 10°C min⫺1 to 250°C, and hold at 250°C for
10 min. Monosaccharides were identified through comparison with the
retention times obtained with individual sugar standards and a composite
standard consisting of Rib, Fuc, Man, Gal, Glc, Ara, Rha, Xyl, GlcUA, and
GalUA.
Positional Cloning of MUM4 and
Determination of cDNA Sequence
A mapping population of 519 plants derived from a cross between
mum4-1 and wild-type Ler was used for progressive fine mapping using
simple sequence length polymorphism and cleaved-amplified polymorphic
sequence markers generated from sequence information provided by the
Arabidopsis Genome Initiative (2000) and Cereon (Jander et al., 2002).
Genomic DNA was isolated from the T-DNA-tagged mum4-3 mutant and
wild-type Wassilewskija plants, and Southern hybridization was performed
using radiolabeled probes derived from F12M16 and T3F20. An insertion
was detected in a 9.5-kb XhoI fragment from T3F20. Gene-specific primers
for the four predicted genes on that fragment were used in PCR with a
T-DNA right border primer (KF-RB p1 5⬘-gttgaagttggcgagttcgt-3⬘). An insertion was identified in At1g53500 using a 3⬘ primer (At1g53500 p2
5⬘-tctgaaactgcctaggtggaa-3⬘).
To confirm that the gene was MUM4, mum4-1 and mum4-2 were sequenced. Sequencing of the Salk Institute sequence-indexed Arabidopsis
T-DNA insertion lines 085051 and 038898 revealed two further alleles,
named mum4-4 and mum4-5, respectively. Molecular complementation of
MUM4 was carried out using a 5.2-kb SalI/BstZ17I fragment of T3F20,
including At1g53500 plus 2.1 kb upstream and 0.4 kb downstream sequence,
into pGREEN0229 (Hellens et al., 2000). mum4-1 plants were transformed
either with the genomic clone (pMUM4g) or the empty vector
(pGREEN0229) using the Agrobacterium tumefaciens dipping method (Clough
and Bent, 1998). Basta-resistant transformants were isolated by spraying
young seedlings with 0.1% (v/v) glufosinate (Final EV150, AgrEvo EH,
Paris) in 0.1% (v/v) Silwet l-77, and putative transformants were verified by
PCR analysis.
The 5⬘-UTR of MUM4 was located using RT-PCR with primers located up
to 900 bp upstream from the predicted ATG in combination with an internal
primer. RNA was isolated from developing siliques according to the protocol of Downing et al. (1992), with the addition of a sodium acetate wash
to remove excess polysaccharides. One-microgram samples were treated
with DNaseI (Invitrogen Life Technologies, Carlsbad, CA) and reverse
transcribed with SuperScript II Reverse Transcriptase (Invitrogen) according to the manufacturer’s instructions. RT-PCR using two sequential primers identified bands approximately 200 bp smaller than predicted from
genomic sequence. The longer fragment was isolated and sequenced.
RHM3 was done with 30 cycles. Semiquantitative RT-PCR (for AP2, TTG1,
TTG2, GL2, and MYB61) was performed a minimum of four times for each
set of primers using two to three different cycle numbers (from 25–30 cycles)
to confirm results.
Accession Numbers
The GenBank accession numbers for genes mentioned in this article are
AY328518 (MUM4 full-length cDNA), AY042833 (RHM1), and AY078958
(RHM3). mum4-3 has been submitted to ABRC under the seed stock number
CS6382.
Distribution of Materials
Upon request, all novel materials described in this publication will be
made available in a timely manner for noncommercial research purposes,
subject to the requisite permission from any third party owners of all or
parts of the material. Obtaining permissions will be the responsibility of the
requestor.
Received October 30, 2003; returned for revision November 12, 2003; accepted November 20, 2003.
ACKNOWLEDGMENTS
We thank Ms. Yeen Ting Hwang (University of British Columbia, Vancouver, BC Canada) and Mr. Adrian Wladichuk (University of British Columbia, Vancouver, BC Canada) for technical assistance, Dr. Michael Bevan
(John Innes Centre, Norwich, UK) for supplying myb61-1 seed, Dr. Mark
Smith (University of British Columbia, Vancouver, BC Canada) and Ms.
Michelle Fawcett (Carnegie Institution, Stanford University, CA) for help
with chemical analysis of mucilage, Drs. Andrew Staehelin (University of
Colorado, Boulder) and Paul Knox (University of Leeds, Leeds, UK) for gifts
of antibodies, and the Salk Institute Genomic Analysis Laboratory (La Jolla,
CA, USA) for providing the sequence-indexed Arabidopsis T-DNA insertion mutants. We also thank Drs. Markus Pauly (Max-Planck Institute for
Molecular Plant Physiology, Golm, Germany) and Björn Usadel (MaxPlanck Institute for Molecular Plant Physiology, Glom, Germany) for communicating unpublished data and delaying publication of their results on
RHM2. We thank members of the Ljerka Haughn (University of British
Columbia, Vancouver, BC Canada) and George Kunst (University of British
Columbia, Vancouver, BC Canada) labs for comments on the manuscript.
LITERATURE CITED
Analyses of Gene Expression
RNA was isolated as described above. For RNA gel blots, 10 ␮g of total
RNA was separated on formaldehyde gels, blotted to Hybond-XL nylon
membranes (Amersham Biosciences Corp., Baie d’Urfé, QC, Canada), and
hybridized according to the manufacturer’s instructions. A 32P-labeled
MUM4 probe corresponding to the degenerate region between the two
predicted enzymatic domains was synthesized using PCR (MUM4p11 5⬘ggaagactttctgatggatctagt-3⬘/MUM4p12 5⬘-gatcaaaaacttcaacgaagc-3⬘). 25S
rRNA bands from the ethidium bromide-stained RNA gel were used as
loading controls.
RNA isolation and RT of DNase-treated RNA was performed as described above. Gene-specific primers surrounding an intron were designed
for each gene, with the exception of TTG1, which lacks an intron. Primers
were as follows: MUM4, p1-ttcgtgtaaatgtcgcaggt/p2 5⬘-gagaagctcgtctagtacggtc-3⬘; RHM1, p1 5⬘-gagactatccgtgccaatgta-3⬘/p2 5⬘-taataactcgtccaacacagtc-3⬘; RHM3, p1 5⬘-ccgagtcaacgttgctgga-3⬘/p2 5⬘-ggtaagagttcatctagtatggtc-3⬘; GAPC, p1 5⬘-tcagactcgagaaagctgctac-3⬘/p2 5⬘-gatcaagtcgaccacacgg3⬘; MYB61, p3 5⬘-tggggagacattcttgctg-3⬘/p4 5⬘-gatgggcttgtgtgtgtttg-3⬘; GL2,
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for each RT reaction. Saturating, qualitative RT-PCR for MUM4, RHM1, and
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